EP3234605B1 - Biosensor auf basis eines gebundenen teilchens - Google Patents

Biosensor auf basis eines gebundenen teilchens Download PDF

Info

Publication number
EP3234605B1
EP3234605B1 EP15808682.7A EP15808682A EP3234605B1 EP 3234605 B1 EP3234605 B1 EP 3234605B1 EP 15808682 A EP15808682 A EP 15808682A EP 3234605 B1 EP3234605 B1 EP 3234605B1
Authority
EP
European Patent Office
Prior art keywords
particle
analyte
moiety
particles
functionalized
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP15808682.7A
Other languages
English (en)
French (fr)
Other versions
EP3234605A1 (de
Inventor
Menno Willem José PRINS
Maarten Merkx
Leonardus Josephus Ijzendoorn
Peter Zijlstra
Emilius Willem Adriaan Visser
Max Rose-Marie Wilhelmus SCHEEPERS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eindhoven Technical University
Original Assignee
Eindhoven Technical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eindhoven Technical University filed Critical Eindhoven Technical University
Priority to PL15808682T priority Critical patent/PL3234605T3/pl
Priority to DK19181414.4T priority patent/DK3567117T3/da
Priority to EP19181414.4A priority patent/EP3567117B1/de
Publication of EP3234605A1 publication Critical patent/EP3234605A1/de
Application granted granted Critical
Publication of EP3234605B1 publication Critical patent/EP3234605B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4412—Scattering spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/149—Particles, e.g. beads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited

Definitions

  • WO2009061783 (A2 ) in the name of University of Rochester describes a sensor chip and detection device.
  • the sensor chip includes a substrate, at least a portion of which is covered by a metal nanoparticle film; a first nucleic acid molecule that is characterized by being able to (i) self-anneal into a hairpin conformation and (ii) hybridize specifically to a target nucleic acid molecule, the first nucleic acid molecule having first and second ends, which first end is tethered to the metal nanoparticle film; and a first fluorophore bound to the second end of the first nucleic molecule.
  • the present invention provides a biosensing technique based on detecting changes in motion of a functionalized particle attached by a tether to a surface, where the presence of an analyte changes the particle between bound and unbound states to the surface.
  • the analyte causes the tethered particle to change between bound and unbound states with the surface. This is a surprising contrast with conventional wisdom in tethered particle motion studies that explicitly teach that binding to the surface is to be avoided.
  • target analytes 110 with two epitopes may be measured with antibodies or fragments thereof coupled to the particle 104 and to the substrate 100.
  • antibody detection can be enabled when moiety 106 and moiety 108 represent antibody-specific epitopes (e.g., peptide epitopes, mimitopes) or native proteins, so that the antibody can form a sandwich complex ( Fig. 2B ) by the binding of its two antigen binding domains to moiety 106 and moiety 108.
  • binding of the molecular pair (moiety 106 and moiety 108) can be induced by analyte (as metal binding between two metal binding domains, or ligand induced cofactor recruitment in nuclear receptor ligand binding domains).
  • target analytes 110 may be measured via steric hindrance, where the binding of target to the particle and/or the substrate changes the parameter space that is accessible by the particle.
  • detection of an analyte may also be performed indirectly through a cascade of intermediate reactions.
  • an enzyme reacts with the analyte and generates a product, which reacts with a biochemical moiety on the particle and/or the substrate surface, and thereby changes a coordinate parameter of the colloido-molecular particle-tether-surface system.
  • the detection of an analyte may be direct or indirect, and in the case of an indirect detection, the analyte-generated product plays the role of the analyte being detected.
  • Embodiments of the invention will allow for the real-time probing of analyte concentrations in complex fluids (e.g., blood, saliva, interstitial skin fluid).
  • complex fluids e.g., blood, saliva, interstitial skin fluid.
  • Single-molecule resolution should be achievable, for high analytical sensitivity.
  • high specificity may be reached by isolating specific from the non-specific interactions. This may allow direct real-time series of measurements in complex fluids, ideally without repeated sample taking or intermediate filtering steps.
  • the sensing system may be used to perform continuous analyte monitoring such as glucose monitoring, e.g., in a competitive format with a glucose binding protein on the particle or on the substrate (e.g., an apo-glucose oxidase, or a periplasmic transport protein, or another glucose-binding molecule).
  • continuous analyte monitoring such as glucose monitoring, e.g., in a competitive format with a glucose binding protein on the particle or on the substrate (e.g., an apo-glucose oxidase, or a periplasmic transport protein, or another glucose-binding molecule).
  • biosensing technique is also relevant for in-vitro diagnostic testing, particularly for point-of-care testing, where it is advantageous if a specific molecular binding process leads to a signal that is detectable by optical means, with little further chemical/biochemical/fluidic processing.
  • a narrowband and coherent light source such as a laser.
  • the high coherence and low bandwidth (typically ⁇ 1 nm) allow for the tight focusing of the beam to achieve a high irradiance of the sample.
  • coherent laser irradiation has limitations because (1) interference fringes cause an inhomogeneous illumination pattern and (2) small spurious reflections and leakage of light in the optical setup cause background artefacts in the image. Such artefacts significantly reduce the signal-to-noise ratio and may fluctuate in time due to vibrations and thermal drifts of the optical setup.
  • White-light sources or lasers are known to be useful to measure spectral shifts.
  • White-light sources exhibit a spectral breadth B much larger than the line width ⁇ of a nanoparticle (i.e., B >> ⁇ ), and thus allows for the measurement of the whole spectrum at once using a spectrometer. Shifts of the spectrum are then extracted by analyzing subsequent spectra.
  • spectral shifts are also measured using a source that is much narrower than the line width of the particle, e.g., a laser (B ⁇ ⁇ ). The time-dependent scattered signal will change when the spectrum of the object shifts.
  • SLDs superluminescent diodes
  • Optical coherence tomography also uses SLDs for illumination, but there is no intention to measure spectral changes of the sample.
  • particles with different plasmon resonances are used, the resonance wavelength is fixed and does not change in time.
  • the measurement of dynamic behavior of plasmons using an SLD is unexpected because it is not intuitive to choose for a light-source that has a bandwidth only slightly narrower than the line width of the resonance that is probed.
  • Instances of the present disclosure include a system and technique for biosensing an analyte in a matrix using a large collection of nanoscale detectors whose optical properties are individually altered in the presence of an analyte.
  • a plasmonic biosensor based on hundreds of individual gold tethered nanoparticles with single-molecule sensitivity are simultaneously monitored in real-time within a dark-field microscopy setup.
  • Spectral signals can be used to identify unreliable objects caused e.g., by particle properties (e.g., shape) or particle configuration (e.g., clusters of particles).
  • particle properties e.g., shape
  • particle configuration e.g., clusters of particles.
  • Individual tethered nanoparticles are characterized by a single narrow Lorentzian spectrum, allowing us to discard clusters based on the line shape and line width of the spectrum. Also, the scattering spectrum of clusters of nanoparticles exhibit a double peak or no clear peak at all. These clusters can easily be distinguished from the spectra of individual particles and are discarded from the data analysis.
  • the temperature of the particles is crucial because the structure and activity of the protein can be impaired when it is heated for extended periods of time.
  • Most globular proteins exhibit a melting temperature ranging from 40°C to 80°C depending on pH and buffer conditions. Based on a theoretical model, it is estimated that an incident intensity exceeding 10 kWcm -2 is needed to raise the particle temperature by more than 10 K.
  • tethered particle motion (TPM) systems may be formed from a 50 nm long double-stranded DNA (dsDNA) tether that attaches a nanoparticle (e.g., microsphere or nanobead) of radius 500 nm to a substrate.
  • dsDNA long double-stranded DNA
  • nanoparticle e.g., microsphere or nanobead
  • the in-plane motion of this particle is tracked in time, so that a two-dimensional projection of the movement of the bead is obtained.
  • Instances of the disclosure may generally use any of various types of polymer (tether) with one end attached to a surface (substrate) and with the other end that is attached to an otherwise free bead (particle).
  • the tethers may be, for example, double-stranded DNA, single-stranded DNA, or RNA, or a polypeptide, or another polymer.
  • any polymer - or even, any macromolecule - that is able to attach a particle to a surface could in principle be used for TPM.
  • TPM systems of the present disclosure use double-stranded DNA (dsDNA) having a persistence length of approximately 50 nm.
  • the size of the particle is preferably less than a few micrometers.
  • Several types of particle may be used, including metal particles (e.g. gold), particles of organic or inorganic material, polystyrene particles, and fluorospheres. Comparing metal particles to polystyrene particles, an advantage of metal particles is the strong scattering of light. On the other hand, polystyrene particles are frequently used in optical tweezer experiments and enable magnetic control of the particle when including a magnetic core.
  • a larger particle has the advantage of higher optical signal and lower motion blur (so higher localization accuracy).
  • a smaller particle has the advantage of a higher diffusivity and more rapid motion, for a higher interaction rate between particle and surface, and so that the change in mobility due to an analyte mediated bond has a higher time resolution.
  • Useful particle sizes are in the range between a few nanometers to a few micrometers, e.g., 5 nm to 10 micrometers.
  • magnetic particles are used that consist of a polymer matrix with many small magnetic iron oxide (Fe 2 O 3 ) grains. Magnetic particles can be actuated with a magnet and can be detected with standard light microscopy.
  • Binding moieties that may be used in the biosensor are e.g., proteins, antibodies, antibody fragments, recombinant proteins, saccharides, molecularly imprinted polymers, small molecules, nucleic acids, DNA, aptamers, multivalent binders, and combinations thereof.
  • the optimal density of binding moieties on particle and substrate depends on the type of assay and on the concentration of analyte. For example, in a sandwich assay with very low analyte concentrations, it is beneficial to have a high density of binding moieties on particle and substrate, in order to have good kinetics and a good sensitivity.
  • a high density may be e.g., between 10 3 and 10 5 moieties/ ⁇ m 2 .
  • at least one of the respective binding moieties should have a low surface density in order to avoid too strong multivalent binding between particle and surface, otherwise the analyte molecules cannot effectively displace the bindings in order to bring the particle in an unbound state.
  • FIG. 9A An apparatus for particle motion measurement according to one embodiment is illustrated in Fig. 9A .
  • Particles 900 are tethered to a substrate by 40 nm dsDNA tethers 902.
  • the molecular tethers are functionalized on one end with biotin for binding to streptavidin 901 that is coated on particles 900, and on the other end with Texas Red 906 for binding to surface-coupled anti-Texas Red antibodies 904.
  • Particle motion is recorded in a dark-field microscopy arrangement, with a white light source 908 and CCD camera 910.
  • the imaging apparatus include a beam block 912 and lenses 914, 916, 918. Images captured by camera 910 are analyzed by processor 911.
  • a series of images 920 are collected, as shown in Fig. 9B , and analyzed to locate particles in every frame.
  • the corresponding motion pattern is constructed as a dot plot 922.
  • the short tether 902 keeps the particle 900 close to the substrate 905 and thereby ensures highfrequency sampling of the particle-substrate interaction.
  • a variety of motion patterns may be resolved with a localization accuracy of a few nanometers.
  • the deviant motion patterns are associated with variations in the number and orientation of the tethers, particle roughness and bindings between particle and surface.
  • PS particles with a diameter of 500 nm were functionalized with antibodies against biotin.
  • the particles were functionalized with 120bp dsDNA using the same protocol as for the MyOne particles (see above). Sedimentation of the PS particles is slower because the mass density is lower and the diameter is smaller than of the MyOne particles. Therefore, an incubation time of 15 minutes was used for the PS particles.
  • the step size is averaged over a window.
  • the standard deviation of the step size in each window is calculated.
  • the amount of fluctuations decreases as expected.
  • increasing the window size also leads to a decrease in the time resolution. There is thus a trade-off between amount of statistical fluctuations and time resolution.
  • the preferred averaging time-window depends e.g., on the size of the particle. E.g., for a 1 micrometer diameter particle, an averaging-window size of 0.1-10 s is preferable.
  • the time interval over which the step size is calculated is varied.
  • the motion is not limited by the time interval over which the step size is calculated, but it is limited by the confinement of the freedom of motion due to the molecular bond.
  • a contrast in the step size is defined as the average step size in the free state divided by the average step size in the bound state, as a function of the time interval ⁇ t. The contrast increases as a function of the ⁇ t, but the time resolution will decrease with ⁇ t. The contrast will converge to a maximum value, as the particle in the free state is limited by the tether.
  • the motion of a particle is more strongly confined compared to the motion of a particle is in the free state.
  • Both the step size of the particle and the area that the particle probes decrease in the bound state.
  • the convex hull is calculated for a shifting window, as shown in Fig. 11B . This is called the area function.
  • the area function may be calculated for a range of window sizes. As the window size is increased from a fraction of a second to several seconds, the area is calculated over more data points and the area function in the free state increases. However, when the system is in the bound state, the area increases significantly less.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Claims (14)

  1. Verfahren zum Erfassen eines Analyten unter Verwendung der Bewegung eines gebundenen Teilchens, wobei das Verfahren Folgendes umfasst:
    Bringen einer Matrix, die den Analyten enthält, in Kontakt mit einer Sensorvorrichtung, die eine Oberfläche und ein Anbindungsmolekül, das an einem ersten Ende an die Oberfläche gebunden ist und an einem zweiten Ende an ein funktionalisiertes Teilchen gebunden ist, aufweist,
    wobei das funktionalisierte Teilchen mit einer ersten Gruppe funktionalisiert ist und die Oberfläche mit einer zweiten Gruppe funktionalisiert ist, so dass das funktionalisierte Teilchen einen ersten Zustand, in dem das funktionalisierte Teilchen an die Oberfläche über die erste Gruppe und die zweite Gruppe gebunden ist, und einen zweiten Zustand aufweist, in dem das funktionalisierte Teilchen nicht an die Oberfläche über die erste Gruppe und die zweite Gruppe gebunden ist, wobei das funktionalisierte Teilchen zwischen dem ersten und dem zweiten Zustand abhängig von der Anwesenheit und Abwesenheit des Analyten wechselt,
    dadurch Verändern der Bewegungscharakteristik des funktionalisierten Teilchens abhängig von der Anwesenheit des Analyten;
    Messen eines Raumkoordinatenparameters des funktionalisierten Teilchens relativ zur Oberfläche; und
    Ermitteln der Anwesenheit/Konzentration des Analyten aus Veränderungen des gemessenen Raumkoordinatenparameters.
  2. Verfahren nach Anspruch 1, wobei das Messen des Raumkoordinatenparameters Folgendes umfasst:
    Beleuchten des funktionalisierten Teilchens und/oder der Oberfläche;
    Erfassen optischer Strahlung von dem funktionalisierten Teilchen und/oder der Oberfläche;
    Ermitteln aus der optischen Strahlung der Position, Orientierung und/oder Geschwindigkeit des funktionalisierten Teilchens in Bezug auf die Oberfläche.
  3. Verfahren nach Anspruch 1, wobei das Messen des Raumkoordinatenparameters Folgendes umfasst:
    Anregen freier Ladungsträger in dem funktionalisierten Teilchen und/oder der Oberfläche;
    Erfassen optischer Strahlung von dem funktionalisierten Teilchen und/oder der Oberfläche;
    wobei das Anregen und/oder Erfassen bei einer Wellenlänge nahe einer Plasmonresonanz des funktionalisierten Teilchens und/oder der Oberfläche durchgeführt wird;
    wobei das Ermitteln der Anwesenheit/Konzentration des Analyten Folgendes umfasst:
    Ermitteln von Veränderungen der erfassten optischen Strahlung.
  4. Verfahren nach Anspruch 1, wobei das Ermitteln der Anwesenheit/Konzentration des Analyten aus Veränderungen des gemessenen Raumkoordinatenparameters das Ermitteln der Anwesenheit/Konzentration des Analyten aus Veränderungen in einer Verteilung von Teilchenlokalisierungen, einer Veränderung des Bereichs eines Musters von Lokalisierungen oder einer Veränderung der Schrittgrößen zwischen Teilchenlokalisierungen umfasst.
  5. Verfahren nach Anspruch 1, wobei das Messen des Raumkoordinatenparameters das Beleuchten des funktionalisierten Teilchens und/oder der Oberfläche von einer Lichtquelle mit einer Linienbreite größer als 5 nm aus oder von einer Superlumineszenzdiode aus umfasst.
  6. Verfahren nach Anspruch 1, wobei das Ermitteln der Anwesenheit/Konzentration des Analyten das Ausführen eines Histogramms und/oder eine Histogrammverarbeitung, um Untergrundrauschen zu unterdrücken und die Spezifität zu erhöhen, umfasst.
  7. Verfahren nach Anspruch 1, wobei das Messen eines Raumkoordinatenparameters des funktionalisierten Teilchens das Messen einer Position des Teilchens, einer Orientierung des Teilchens, einer Winkelgeschwindigkeit des Teilchens oder einer linearen Geschwindigkeit des Teilchens umfasst.
  8. Biosensor zum Erfassen eines Analyten unter Verwendung der Bewegung eines gebundenen Teilchens, wobei der Biosensor Folgendes umfasst:
    ein Teilchen;
    eine Oberfläche;
    ein Anbindungsmolekül, das an einem ersten Ende an das Teilchen gebunden ist und an einem zweiten Ende an die Oberfläche gebunden ist;
    eine erste Gruppe, die an das Teilchen oder an das Anbindungsmolekül konjugiert ist;
    eine zweite Gruppe, die an die Oberfläche oder an das Anbindungsmolekül konjugiert ist;
    wobei, falls die erste Gruppe an das Anbindungsmolekül konjugiert ist, die zweite Gruppe an die Oberfläche konjugiert ist,
    und falls die zweite Gruppe an das Anbindungsmolekül konjugiert ist, die erste Gruppe an das Teilchen konjugiert ist;
    wobei die erste Gruppe und die zweite Gruppe eine Bindungsaffinität zueinander abhängig von der Anwesenheit oder Abwesenheit eines Zielanalyten aufweisen oder eine Bindungsaffinität zum Zielanalyten aufweisen, wobei sich eine Bewegungscharakteristik des Teilchens abhängig von der Anwesenheit des Analyten verändert, wodurch es ermöglicht wird, den Analyten durch Messen der Veränderungen eines Raumkoordinatenparameters des Teilchens in Bezug auf die Oberfläche zu erfassen.
  9. Biosensor nach Anspruch 8, wobei die erste Gruppe an das Anbindungsmolekül an einem Punkt konjugiert ist, der nahe dem ersten Ende des Anbindungsmoleküls liegt.
  10. Biosensor nach Anspruch 8, wobei die zweite Gruppe an das Anbindungsmolekül an einem Punkt konjugiert ist, der nahe dem zweiten Ende des Anbindungsmoleküls liegt.
  11. Biosensor nach Anspruch 8, wobei der Biosensor einen Bindungsassay, einen kompetitiven Assay, einen Verdrängungsassay, einen Sandwich-Assay, einen enzymatischen Assay, einen Assay mit Zielamplifikation und/oder Signalverstärkung, einen mehrstufigen Assay oder einen Assay mit molekularer Kaskade ausführt.
  12. Biosensor nach Anspruch 8, wobei die erste Gruppe oder die zweite Gruppe ein Protein, ein Antikörper, ein Fragment davon, ein rekombinantes Protein, ein Saccharid, ein molekular geprägtes Polymer, ein kleines Molekül, eine Nukleinsäure, ein DNA-Molekül, ein Aptamer, ein multivalenter Binder oder eine Kombination davon ist.
  13. Biosensor nach Anspruch 8, wobei die erste Gruppe oder die zweite Gruppe ein Glucose-bindendes Molekül ist.
  14. Biosensor nach Anspruch 8, angepasst, um Multiplexen, Analyten-Multiplexen, Raum-Multiplexen, Multiplexen in Position, Teilchen-basiertes Multiplexen, Verwendung von Teilchen mit unterschiedlichen optischen Eigenschaften, Verwendung mehrerer Wellenlängen, spektroskopisches Multiplexen, Multiplexen mit Partikeln, die unterschiedliche Rezeptoren auf ihrer Oberfläche haben, Sondenfunktion-Multiplexen, Verwendung unterschiedlicher Sondenfunktionalisierungen, Verwendung verschiedener Sondenbeschichtungen, Verwendung verschiedener Sondenabdeckungen oder Kombinationen davon auszuführen.
EP15808682.7A 2014-12-16 2015-12-15 Biosensor auf basis eines gebundenen teilchens Active EP3234605B1 (de)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PL15808682T PL3234605T3 (pl) 2014-12-16 2015-12-15 Bioczujnik na bazie cząstki na uwięzi
DK19181414.4T DK3567117T3 (da) 2014-12-16 2015-12-15 Biosensor baseret på en bundet partikel
EP19181414.4A EP3567117B1 (de) 2014-12-16 2015-12-15 Biosensor auf basis eines gebundenen teilchens

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201462092751P 2014-12-16 2014-12-16
US201462092763P 2014-12-16 2014-12-16
US201562132096P 2015-03-12 2015-03-12
PCT/EP2015/079864 WO2016096901A1 (en) 2014-12-16 2015-12-15 Biosensor based on a tethered particle

Related Child Applications (1)

Application Number Title Priority Date Filing Date
EP19181414.4A Division EP3567117B1 (de) 2014-12-16 2015-12-15 Biosensor auf basis eines gebundenen teilchens

Publications (2)

Publication Number Publication Date
EP3234605A1 EP3234605A1 (de) 2017-10-25
EP3234605B1 true EP3234605B1 (de) 2019-07-03

Family

ID=54849954

Family Applications (2)

Application Number Title Priority Date Filing Date
EP19181414.4A Active EP3567117B1 (de) 2014-12-16 2015-12-15 Biosensor auf basis eines gebundenen teilchens
EP15808682.7A Active EP3234605B1 (de) 2014-12-16 2015-12-15 Biosensor auf basis eines gebundenen teilchens

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP19181414.4A Active EP3567117B1 (de) 2014-12-16 2015-12-15 Biosensor auf basis eines gebundenen teilchens

Country Status (8)

Country Link
US (2) US10519486B2 (de)
EP (2) EP3567117B1 (de)
JP (1) JP6776240B2 (de)
CN (2) CN107209178B (de)
DK (1) DK3567117T3 (de)
ES (1) ES2743703T3 (de)
PL (1) PL3234605T3 (de)
WO (1) WO2016096901A1 (de)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9627114B2 (en) * 2015-09-14 2017-04-18 Elwha Llc Magnetic plasmonic nanoparticle positioned on a magnetic plasmonic substrate
US10768406B2 (en) * 2016-06-20 2020-09-08 Scanogen Inc. Imaging system
DK3563157T3 (da) 2016-12-28 2022-10-24 Univ Eindhoven Tech Bundet-partikelbiosensor med forskellige fangstmolekyler til påvisning af omkoblingshændelser
EP3574325B1 (de) * 2017-03-03 2023-11-08 Technische Universiteit Eindhoven Biosensor mit gap-region für kontinuierliche überwachung
GB2565074A (en) * 2017-07-31 2019-02-06 Univ Bristol Method and apparatus for bacterial analysis
GB201807409D0 (en) * 2018-05-04 2018-06-20 Oxford Nanoimaging Ltd Assay
TWI664397B (zh) * 2018-07-10 2019-07-01 精準基因生物科技股份有限公司 感測裝置
GB201818421D0 (en) * 2018-11-12 2018-12-26 Cambridge Entpr Ltd Magnetic particle and method
JP7246085B2 (ja) * 2019-08-07 2023-03-27 国立研究開発法人産業技術総合研究所 標的物質検出装置
CN111366563B (zh) * 2020-03-13 2022-04-19 量准（上海）医疗器械有限公司 数字化等离子免疫吸附试剂盒及其制造和测试方法
EP4200591A1 (de) 2020-08-21 2023-06-28 Technische Universiteit Eindhoven Biosensor mit partikelbewegung
KR102522280B1 (ko) * 2020-09-25 2023-04-18 연세대학교 산학협력단 매미날개구조 생체모방 나노스파이크 충돌판이 적용된 전기적 임팩터
US11953437B2 (en) 2021-08-25 2024-04-09 Endress+Hauser Optical Analysis, Inc. Device and method for measuring multiple analyte concentrations in a measuring medium
US20230182127A1 (en) * 2021-12-10 2023-06-15 Bio-Rad Laboratories, Inc. Compositions, methods, and systems for sample processing with morphology-adjustable functionalized particles

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002211587A1 (en) * 2000-10-10 2002-04-22 Bioforce Nanosciences, Inc Nanoscale sensor
WO2003029435A2 (en) 2001-10-04 2003-04-10 The Regents Of The University Of California Detection of polynucleotide hybridization
US7323347B2 (en) 2004-05-27 2008-01-29 Sensata Technologies, Inc. Biosensor surface structures and methods
CN101573618A (zh) * 2006-09-15 2009-11-04 赫摩耐提克斯公司 通过光学操作与功能化表面有关的颗粒进行表面作图
WO2009061783A2 (en) 2007-11-05 2009-05-14 University Of Rochester Dna microarray having hairpin probes tethered to nanostructured metal surface
US20120184048A1 (en) 2009-09-28 2012-07-19 Koninklijke Philips Electronics N.V. Method for characterization of biological bonds
WO2011053894A2 (en) 2009-11-01 2011-05-05 Caerus Molecular Diagnostics Incorporated Methods and apparatus for binding assays
US20120288852A1 (en) * 2010-01-15 2012-11-15 Richard Willson Force Mediated Assays
EP2388337B1 (de) 2010-04-30 2014-07-02 Nxp B.V. Messgerät und Herstellungsverfahren dafür
EP3508853A1 (de) * 2014-11-12 2019-07-10 Technische Universiteit Eindhoven Biosensor mit dynamischer umschaltung

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
CN107209178B (zh) 2020-04-21
EP3567117A1 (de) 2019-11-13
EP3234605A1 (de) 2017-10-25
US11359231B2 (en) 2022-06-14
CN111381031B (zh) 2023-09-29
EP3567117B1 (de) 2022-02-23
CN111381031A (zh) 2020-07-07
JP2018500559A (ja) 2018-01-11
CN107209178A (zh) 2017-09-26
ES2743703T3 (es) 2020-02-20
JP6776240B2 (ja) 2020-10-28
US10519486B2 (en) 2019-12-31
WO2016096901A1 (en) 2016-06-23
US20170362645A1 (en) 2017-12-21
PL3234605T3 (pl) 2019-12-31
US20200140932A1 (en) 2020-05-07
DK3567117T3 (da) 2022-05-30

Similar Documents

Publication Publication Date Title
US11359231B2 (en) Biosensor based on a tethered particle
US10330676B2 (en) Plasmonic biosensor based on molecular conformation
US10603650B2 (en) Multiplexed surface enhanced Raman sensors for early disease detection and in-situ bacterial monitoring
US10656149B2 (en) Analyte detection enhancement by targeted immobilization, surface amplification, and pixelated reading and analysis
EP3218717B1 (de) Biosensor mit dynamischer umschaltung
WO2014144133A1 (en) Analyte detection enhancement by targeted immobilization, surface amplification, and pixelated reading and analysis
JP2017538917A (ja) Ｌｓｐｒセンサを取り込むモバイル／装着式デバイス
KR20160138059A (ko) 향상된 검정 감도를 위한 디지털 lspr
US9993185B2 (en) Plasmonics nanostructures for multiplexing implantable sensors
US10001442B2 (en) Optical fiber-based hybrid SERS platform for in vivo detection of bio-molecules
Yeung et al. Multiplex detection of urinary miRNA biomarkers by transmission surface plasmon resonance
KR101470730B1 (ko) 파장-의존성 플라즈몬 공명산란을 이용한 나노바이오칩 키트 및 이를 이용한 생체분자의 검출방법
WO2011154918A2 (en) Diagnostic apparatus for immunoassay and diagnostic method for immunoassay using the same
US20130224768A1 (en) Immunochromatographic assay method and apparatus
WO2016096908A1 (en) Biosensor based on single-molecule fluorescence detection
Meenakshi et al. 10 SERS Analysis for Single
Meenakshi et al. SERS Analysis for Single-Molecule Detection of Disease Biomarkers
Wang et al. Conference 6380: Smart Medical and Biomedical Sensor Technology IV

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20170616

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20180507

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20190325

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

Ref country code: AT

Ref legal event code: REF

Ref document number: 1151620

Country of ref document: AT

Kind code of ref document: T

Effective date: 20190715

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602015033240

Country of ref document: DE

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: NL

Ref legal event code: FP

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: STOLMAR AND PARTNER INTELLECTUAL PROPERTY S.A., CH

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1151620

Country of ref document: AT

Kind code of ref document: T

Effective date: 20190703

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191003

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191104

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191003

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191004

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191103

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200224

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602015033240

Country of ref document: DE

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG2D Information on lapse in contracting state deleted

Ref country code: IS

26N No opposition filed

Effective date: 20200603

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20191231

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20191215

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20191215

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20191231

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20151215

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190703

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: TR

Payment date: 20221215

Year of fee payment: 8

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230731

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20231220

Year of fee payment: 9

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 20231220

Year of fee payment: 9

Ref country code: NL

Payment date: 20231220

Year of fee payment: 9

Ref country code: IT

Payment date: 20231228

Year of fee payment: 9

Ref country code: FR

Payment date: 20231221

Year of fee payment: 9

Ref country code: DE

Payment date: 20231214

Year of fee payment: 9

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: PL

Payment date: 20231121

Year of fee payment: 9

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: ES

Payment date: 20240129

Year of fee payment: 9

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20240102

Year of fee payment: 9